Non‐Typeable Haemophilus influenzae Infection of the Junbo Mouse

Michael T. Cheeseman1, Derek W. Hood2

1 Developmental Biology Division, The Roslin Institute and Royal (Dick) School of Veterinary Studies University of Edinburgh, 2 Molecular Genetics Unit, MRC Harwell Institute, Harwell Science and Innovation Campus
Publication Name:  Current Protocols in Mouse Biology
Unit Number:   
DOI:  10.1002/cpmo.24
Online Posting Date:  March, 2017
GO TO THE FULL TEXT: PDF or HTML at Wiley Online Library


Acute otitis media, inflammation of the middle ear bulla, is the most common bacterial infection in children. For one of the principal otopathogens, non‐typeable Haemophilus influenzae (NTHi), animal models allow us to investigate host‐microbial interactions relevant to the onset and progression of infection and to study treatment of middle ear disease. We have established a robust model of NTHi middle ear infection in the Junbo mouse. Intranasal inoculation with NTHi produces high rates of bulla infection and high bacterial titers in bulla fluids; bacteria can also spread down the respiratory tract to the mouse lung. An innate immune response is detected in the bulla of Junbo mice following NTHi infection, and bacteria are maintained in some ears at least up to day 56 post‐inoculation. The Junbo/NTHi infection model facilitates studies on bacterial pathogenesis and antimicrobial intervention regimens and vaccines for better treatment and prevention of NTHi middle ear infection. © 2017 by John Wiley & Sons, Inc.

Keywords: Junbo mouse; lung infection; middle ear infection; non‐typeable Haemophilus influenzae (NTHi); otitis media

PDF or HTML at Wiley Online Library

Table of Contents

  • Introduction
  • Basic Protocol 1: Junbo Mouse NTHi Middle Ear Infection
  • Support Protocol 1: Junbo Mouse NTHi Middle Ear Infection: Histology and Distribution of NTHi
  • Support Protocol 2: Immune Response Measurement by Real‐Time Quantitative PCR (RT‐qPCR) of Bulla Fluids
  • Basic Protocol 2: Mouse Immunization and Protection Against NTHi Infection
  • Basic Protocol 3: Antimicrobial Treatment of NTHi Infection
  • Basic Protocol 4: NTHi Mouse Pulmonary Infection Model
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
PDF or HTML at Wiley Online Library


Basic Protocol 1: Junbo Mouse NTHi Middle Ear Infection

  • NTHi [stored at –80ºC in BHI (see below)/20% (v/v) glycerol]: human OM disease isolates (162, 176, 375, 486, 1124 and 1158; available from MRC Harwell Institute) are used by us for mouse infection studies (Cody et al., ); streptomycin‐resistant NTHi strains are designated sr, e.g., strain 162sr
  • Supplemented brain‐heart infusion (sBHI) broth and agar plates (see recipe)
  • Mice (8 to 11 weeks of age): Junbo mice are congenic on a C3H/HeH background (Parkinson et al., ); mice are housed under SPF conditions and are mostly used at 8 to 11 typically used by us at 8±1 or 11±1 weeks of age with a similar experimental outcome [available from the European Mouse Mutant Archive (EM:00091) via MRC Harwell Institute; for non‐academic groups, available through MRC Technology, U.K.]; for some studies, germ‐free (GF) mice are used (details of the mouse husbandry and microbiological surveillance are found in Hood et al., )
  • Sterile PBS/2% (w/v) gelatin (Fluka, cat. no. 48723) for inoculum preparation
  • Barbiturate solution (50% Euthatal) for intraperitoneal injection (delivered at 3.3 ml/kg body weight); alternatively a rising CO 2 concentration (Donovan and Brown, ) may be used to euthanize mice
  • PBS for collecting and diluting in vivo samples
  • 70% ethanol to sterilize the instruments between the sampling of ears
  • 37ºC, 5% CO 2 incubator
  • 1‐μl inoculating loop
  • Spectrophotometer for determining OD 490
  • Scissors for removal of the head
  • Binocular dissecting microscope with 10× magnification and LED stage lighting.
  • Fine forceps for puncture of the tympanic membrane
  • < 2‐µl‐volume pipet with sterile filtered micro‐tip to collect the small volume of ME fluids
  • Additional reagents and equipment for assessing titer of bacteria (Elbing and Brent, ), isoflurane anesthesia of mice (Phoon and Turnbull, ), injection of mice (Donovan and Brown, ), and euthanasia of mice by CO 2 asphyxiation (Donovan and Brown, )

Support Protocol 1: Junbo Mouse NTHi Middle Ear Infection: Histology and Distribution of NTHi

  • Dissected mouse heads ( protocol 1)
  • Xylene
  • 10% neutral buffered formalin
  • 100% ethanol
  • Tris‐buffered saline (TBS: see recipe)
  • Dako REAL peroxidase blocker (code no. S2023)
  • Dako proteinase K (code no. S3020)
  • Primary antibody [e.g., rat monoclonal anti‐F4/80 (Serotec, cat. no. MCA497G); rabbit polyclonal anti‐caspase 3 (Abcam, cat. no. ab2302); rabbit polyclonal anti‐histone 3 (Abcam, cat. no. ab61251)]
  • ImmPress HRP anti‐rat kit (Vector Labs, cat. no. MP‐744‐15)
  • Dako antibody diluent (code no. S0809)
  • Dako REAL peroxidase blocker (code no. S2023)
  • Dako liquid DAB+ substrate chromogen system (code no. K3468)
  • Harris hematoxylin (Sigma‐Aldrich, cat. no. HH516)
  • Dako Envision+ System HRP anti‐rabbit antibody (code no. K4011)
  • HRP Visualization Kit (Advanced Cell Diagnostics)
  • ClearVue mountant (Thermo Fisher Scientific)
  • Probe B‐HInfluenzae‐NTHi375‐16SrRNA (Advanced Cell Diagnostics)
  • Positive control for RNA integrity (e.g., PpiB; Advanced Cell Diagnostics)
  • Negative hybridization control (e.g., DapB; Advanced Cell Diagnostics)
  • Electrostatically charged slides (e.g., SuperFrost Plus; VWR Scientific)
  • 60ºC drying oven
  • Slide scanner: e.g., Hamamatsu NanoZoomer
  • Software for morphometric measurements (e.g., Hamamatsu NanoZoomer)

Support Protocol 2: Immune Response Measurement by Real‐Time Quantitative PCR (RT‐qPCR) of Bulla Fluids

  Additional Materials (also see protocol 1)
  • Germ‐free Junbo mice (MRC Harwell Institute)
  • RNase‐free H 2O
  • Nucleospin RNA/protein isolation kits (Macherey‐Nagel)
  • AB 7500 software v2.0.1
  • Additional reagents and equipment for quantitative PCR (qPCR; Bookout et al., ; Cheeseman et al., )

Basic Protocol 2: Mouse Immunization and Protection Against NTHi Infection

  • 5‐week‐old SPF Junbo mice (MRC Harwell Institute; see protocol 1 materials list for more information)
  • Live NTHi bacteria ( protocol 1, steps 1 to 4) for intranasal challenge post‐immunization
  • Supplemented brain‐heart infusion (sBHI) broth and agar plates (see recipe)
  • Phosphate‐buffered saline (PBS; see recipe) containing 0.08% (w/v) paraformaldehyde
  • Phosphate‐buffered saline (PBS; see recipe)
  • Adjuvant (Adjuplex from Sigma)
  • 1.1‐ml Z‐Gel spin columns (Sarstedt)
  • Microscope slides
  • Microscope with phase‐contrast optics
  • Additional reagents and equipment for blood collection from mice (Donovan and Brown, ), injection of mice (Donovan and Brown, ), and intranasal inoculation and determination of ME infection rate and bacterial titer by terminal sampling ( protocol 1)

Basic Protocol 3: Antimicrobial Treatment of NTHi Infection

  • 8‐week old SPF Junbo mice (MRC Harwell Institute; see protocol 1 materials list for more information)
  • NTHi bacteria for intranasal challenge as described in protocol 1
  • Azithromycin (Sigma‐Aldrich, cat. no. 57947) in 2% methoxycellulose (or other antibiotic in solution as appropriate)
  • 2% methoxycellulose (Sigma‐Aldrich, cat. no. M7140)
  • Gavage needle
  • Additional reagents and equipment for intranasal inoculation and determination of ME infection rate and bacterial titer by terminal sampling ( protocol 1)

Basic Protocol 4: NTHi Mouse Pulmonary Infection Model

  • NTHi bacteria for intranasal challenge (see protocol 1)
  • 8‐week‐old SPF Junbo mice (see protocol 1 materials list for more information)
  • Homogenizer (we use an IKA Ultra‐Turax T25 operated in a Class II microbiological safety cabinet)
  • Additional reagents and equipment for intranasal inoculation and determination of ME infection rate and bacterial titer by terminal sampling ( protocol 1) and fixation, paraffin embedding, sectioning, and staining ( protocol 2)
PDF or HTML at Wiley Online Library



Literature Cited

Literature Cited
  Bakaletz, L.O. 2009. Chinchilla as a robust, reproducible and polymicrobial model of otitis media and its prevention. Expert Rev. Vaccines. 8:1063‐1082. doi: 10.1586/erv.09.63.
  Bookout, A.L., Cummins, C.L., Mangelsdorf, D.J., Pesola, J.M. and Kramer, M.F. 2006. High‐throughput real‐time quantitative reverse transcription PCR. Curr. Protoc. Mol. Biol. 73:15.8.1‐15.8.28. doi: 10.1002/0471142727.mb1508s73.
  Chang, S.H., Mirabolfathinejad, S.G., Katta, H., Cumpian, A.M., Gong, L., Caetano, M.S., Moghaddam, S.J., and Dong, C. 2014. T helper 17 cells play a critical pathogenic role in lung cancer. Proc. Natl. Acad. Sci. U. S. A. 111:5664‐5669. doi: 10.1073/pnas.1319051111.
  Cheeseman, M.T., Tyrer, H.E., Williams, D., Hough, T.A., Pathak, P., Romero, M.R., Hilton, H., Bali, S., Parker, A., Vizor, L., Purnell, T., Vowell, K., Wells, S., Bhutta, M.F., Potter, P.K., and Brown, S.D. 2011. HIF‐VEGF pathways are critical for chronic otitis media in Junbo and Jeff mouse mutants. PLoS Genet. 7:e1002336. doi: 10.1371/journal.pgen.1002336.
  Clark, J.M., Brinson, G., Newman, M.K., Jewett, B.S., Sartor, B.R., Prazma, J., and Pillsbury, H.C., 3rd. 2000. An animal model for the study of genetic predisposition in the pathogenesis of middle ear inflammation. Laryngoscope 110:1511‐1515. doi: 10.1097/00005537‐200009000‐00019.
  Cody, A.J., Field, D., Feil, E.J., Stringer, S., Deadman, M.E., Tsolaki, A.G., Gratz, B., Bouchet, V., Goldstein, R., Hood, D.W., and Moxon, E.R. 2003. High rates of recombination in otitis media isolates of non‐typeable Haemophilus influenzae. Infect. Genet. Evol. 3:57‐66. doi: 10.1016/S1567‐1348(02)00152‐1.
  De Chiara, M., Hood, D., Muzzi, A., Pickard, D.J., Perkins, T., Pizza, M., Dougan, G., Rappuoli, R., Moxon, E.R., Soriani, M., and Donati, C. 2014. Genome sequencing of disease and carriage isolates of nontypeable Haemophilus influenzae identifies discrete population structure. Proc. Natl. Acad Sci. U. S.A. 111:5439‐5444. doi: 10.1073/pnas.1403353111.
  Donovan, J. and Brown, P. 2006a. Parenteral injections. Curr. Protoc. Immunol. 73:1.6.1‐1.6.10. doi: 10.1002/0471142735.im0106s73.
  Donovan, J. and Brown, P. 2006b. Euthanasia. Curr. Protoc. Immunol. 73:1.8.1‐1.8.4. doi: 10.1002/0471142735.im0108s73.
  Donovan, J. and Brown, P. 2006c. Blood collection. Curr. Protoc. Immunol. 73:1.7.1‐1.7.9. doi: 10.1002/0471142735.im0107s73.
  Elbing, K. and Brent, R. 2002. Growth on solid media. Curr. Protoc. Mol. Biol. 59:1.3.1‐1.3.6. doi: 10.1002/0471142735.im0103s73.
  Ercoli, G., Baddal, B., Alessandra, G., Marchi, S., Petracca, R., Arico, B., Pizza, M., Soriani, M., and Rossi‐Paccani, S. 2015. Development of a serological assay to predict antibody bactericidal activity against non‐typeable Haemophilus influenzae. BMC Microbiol. 15:87. doi: 10.1186/s12866‐015‐0420‐x.
  Euba, B., Moleres, J., Segura, V., Viadas, C., Morey, P., Moranta, D., Leiva, J., de‐Torres, J.P., Bengoechea, J.A., and Garmendia, J. 2015a. Genome expression profiling‐based identification and administration efficacy of host‐directed antimicrobial drugs against respiratory infection by nontypeable Haemophilus influenzae. Antimicrob. Agents Chemother. 59:7581‐7592. doi: 10.1128/AAC.01278‐15.
  Euba, B., Moleres, J., Viadas, C., Ruiz de los Mozos, I., Valle, J., Bengoechea, J.A., and Garmendia, J. 2015b. Relative contribution of P5 and hap surface proteins to nontypable Haemophilus influenzae interplay with the host upper and lower airways. PLoS ONE 10:e0123154. doi: 10.1371/journal.pone.0123154.
  Hardisty‐Hughes, R.E., Tateossian, H., Morse, S.A., Romero, M.R., Middleton, A., Tymowska‐Lalanne, Z., Hunter, A.J., Cheeseman, M., and Brown, S.D. 2006. A mutation in the F‐box gene, Fbxo11, causes otitis media in the Jeff mouse. Hum. Mol. Genet. 15:3273‐3279. doi: 10.1093/hmg/ddl403.
  Hood, D., Moxon, R., Purnell, T., Richter, C., Williams, D., Azar, A., Crompton, M., Wells, S., Fray, M., Brown, S.D., and Cheeseman, M.T. 2016. A new model for non‐typeable Haemophilus influenzae middle ear infection in the Junbo mutant mouse. Dis. Model. Mech. 9:69‐79. doi: 10.1242/dmm.021659.
  Juhn, S.K., Jung, M.K., Hoffman, M.D., Drew, B.R., Preciado, D.A., Sausen, N.J., Jung, T.T., Kim, B.H., Park, S.Y., Lin, J., Ondrey, F.G., Mains, D.R., and Huang, T. 2008. The role of inflammatory mediators in the pathogenesis of otitis media and sequelae. Clin. Exp. Otorhinolaryngol. 1:117‐138. doi: 10.3342/ceo.2008.1.3.117.
  Kaur, R., Casey, J., and Pichichero, M. 2015. Cytokine, chemokine, and Toll‐like receptor expression in middle ear fluids of children with acute otitis media. The Laryngoscope 125:E39‐44. doi: 10.1002/lary.24920.
  Langereis, J.D., Stol, K., Schweda, E.K., Twelkmeyer, B., Bootsma, H.J., de Vries, S.P., Burghout, P., Diavatopoulos, D.A., and Hermans, P.W. 2012. Modified lipooligosaccharide structure protects nontypeable Haemophilus influenzae from IgM‐mediated complement killing in experimental otitis media. mBio 3:e00079‐00012. doi: 10.1128/mBio.00079‐12.
  Lugade, A.A., Bogner, P.N., Thatcher, T.H., Sime, P.J., Phipps, R.P., and Thanavala, Y. 2014. Cigarette smoke exposure exacerbates lung inflammation and compromises immunity to bacterial infection. J. Immunol. 192:5226‐5235. doi: 10.4049/jimmunol.1302584.
  Morey, P., Viadas, C., Euba, B., Hood, D.W., Barberan, M., Gil, C., Grillo, M.J., Bengoechea, J.A., and Garmendia, J. 2013. Relative contributions of lipooligosaccharide inner and outer core modifications to nontypeable Haemophilus influenzae pathogenesis. Infect. Immun. 81:4100‐4111. doi: 10.1128/IAI.00492‐13.
  Murphy, T.F. 2003. Respiratory infections caused by non‐typeable Haemophilus influenzae. Curr. Opin. Infect. Dis. 16:129‐134. doi: 10.1097/00001432‐200304000‐00009.
  Pang, B., Hong, W., West‐Barnette, S.L., Kock, N.D., and Swords, W.E. 2008. Diminished ICAM‐1 expression and impaired pulmonary clearance of nontypeable Haemophilus influenzae in a mouse model of chronic obstructive pulmonary disease/emphysema. Infect. Immun. 76:4959‐4967. doi: 10.1128/IAI.00664‐08.
  Parker, A., Chessum, L., Mburu, P., Sanderson, J., and Bowl, M.R. 2016. Light and electron microscopy methods for examination of cochlear morphology in mouse models of deafness. Curr. Protoc. Mouse Biol. 6:272‐306. doi: 10.1002/cpmo.10
  Parkinson, N., Hardisty‐Hughes, R.E., Tateossian, H., Tsai, H.T., Brooker, D., Morse, S., Lalane, Z., MacKenzie, F., Fray, M., Glenister, P., Woodward, A.M., Polley, S., Barbaric, I., Dear, N., Hough, T.A., Hunter, A.J., Cheeseman, M.T., and Brown, S.D. 2006. Mutation at the Evi1 locus in Junbo mice causes susceptibility to otitis media. PLoS Genet. 2:e149. doi: 10.1371/journal.pgen.0020149.
  Phoon, C.K.L. and Turnbull, D.H. 2016. Cardiovascular imaging in mice. Curr. Protoc. Mouse Biol. 6:15‐38. doi: 10.1002/9780470942390.mo150122
  Roos, A.B., Sethi, S., Nikota, J., Wrona, C.T., Dorrington, M.G., Sanden, C., Bauer, C.M., Shen, P., Bowdish, D., Stevenson, C.S., Erjefalt, J.S., and Stampfli, M.R. 2015. IL‐17A and the promotion of neutrophilia in acute exacerbation of chronic obstructive pulmonary disease. Am. J. Respir. Crit. Care. Med. 192:428‐437. doi: 10.1164/rccm.201409‐1689OC.
  Rye, M.S., Bhutta, M.F., Cheeseman, M.T., Burgner, D., Blackwell, J.M., Brown, S.D., and Jamieson, S.E. 2011. Unraveling the genetics of otitis media: From mouse to human and back again. Mamm. Genome. 22:66‐82. doi: 10.1007/s00335‐010‐9295‐1.
  Sethi, S. and Murphy, T.F. 2001. Bacterial infection in chronic obstructive pulmonary disease in 2000: A state‐of‐the‐art review. Clin. Microbiol. Rev. 14:336‐363. doi: 10.1128/CMR.14.2.336‐363.2001.
  Shann, F., Hart, K., and Thomas, D. 1984. Acute lower respiratory tract infections in children: Possible criteria for selection of patients for antibiotic therapy and hospital admission. Bull. World Health Organ. 62:749‐753.
  Su, Y.C., Mukherjee, O., Singh, B., Hallgren, O., Westergren‐Thorsson, G., Hood, D., and Riesbeck, K. 2016. Haemophilus influenzae P4 interacts with extracellular matrix proteins promoting adhesion and serum resistance. J. Infect. Dis. 213:314‐323. doi: 10.1093/infdis/jiv374.
  Tateossian, H., Morse, S., Parker, A., Mburu, P., Warr, N., Acevedo‐Arozena, A., Cheeseman, M., Wells, S., and Brown, S.D. 2013. Otitis media in the Tgif knockout mouse implicates TGFbeta signalling in chronic middle ear inflammatory disease. Hum. Mol. Genet. 22:2553‐2565. doi: 10.1093/hmg/ddt103.
  Unger, B.L., Faris, A.N., Ganesan, S., Comstock, A.T., Hershenson, M.B., and Sajjan, U.S. 2012. Rhinovirus attenuates non‐typeable Hemophilus influenzae–stimulated IL‐8 responses via TLR2‐dependent degradation of IRAK‐1. PLoS Pathog. 8:e1002969. doi: 10.1371/journal.ppat.1002969.
  Woo, J.I., Oh, S., Webster, P., Lee, Y.J., Lim, D.J., and Moon, S.K. 2014. NOD2/RICK‐dependent beta‐defensin 2 regulation is protective for nontypeable Haemophilus influenzae‐induced middle ear infection. PLoS ONE 9:e90933. doi: 10.1371/journal.pone.0090933.
  Woo, J.I., Kil, S.H., Brough, D.E., Lee, Y.J., Lim, D.J., and Moon, S.K. 2015. Therapeutic potential of adenovirus‐mediated delivery of beta‐defensin 2 for experimental otitis media. Innate. Immun. 21:215‐224. doi: 10.1177/1753425914534002.
  Xu, X., Woo, C.H., Steere, R.R., Lee, B.C., Huang, Y., Wu, J., Pang, J., Lim, J.H., Xu, H., Zhang, W., Konduru, A.S., Yan, C., Cheeseman, M.T., Brown, S.D., and Li, J.D. 2012. EVI1 acts as an inducible negative‐feedback regulator of NF‐kappaB by inhibiting p65 acetylation. J. Immunol. 188:6371‐6380. doi: 10.4049/jimmunol.1103527.
  Yao, W., Frie, M., Pan, J., Pak, K., Webster, N., Wasserman, S.I., and Ryan, A.F. 2014. C‐Jun N‐terminal kinase (JNK) isoforms play differing roles in otitis media. BMC Immunol. 15:46. doi: 10.1186/s12865‐014‐0046‐z.
PDF or HTML at Wiley Online Library